Project Summary/Abstract: The tRNAHis guanylyltransferase (Thg1) is absolutely essential in yeast, and likely throughout all eukaryotes, due to the universal requirement for G-1 on tRNAHis in all eukaryotes in which it has been investigated. Thg1 adds G-1 to tRNAHis via an unusual non-templated 3'-5'nucleotide addition reaction, by an unknown catalytic mechanism that cannot be predicted based on similarity to known enzymes, and thus is likely to employ a novel catalytic mechanism. Moreover, we have recently demonstrated that all Thg1 family members catalyze a template-dependent 3'-5'addition reaction with various substrates, and that this activity is used for a form of G-1 addition in archaea, as well as for an unusual tRNA editing reaction in protozoa. These demonstrated roles for templated 3'-5'addition greatly expand the scope of catalytic activities exhibited by Thg1 family members. Nonetheless, the presence of Thg1 homologs in archaea and bacteria that do not require enzymatic G-1 addition to tRNAHis and unexplained Thg1-related phenotypes in yeast and human cells suggest that additional roles for 3'-5'addition are yet to be uncovered. This application proposes the use of kinetic, genetic, biochemical and structural techniques to investigate the molecular mechanisms and biological functions of both non-templated and templated 3'-5'addition reactions catalyzed by diverse Thg1 family members. These results will provide insight into catalysis of a novel and apparently widespread, but largely unexplored, reaction in biology, and will enable further investigation into alternative functions for 3'-5'nucleotide addition in biological systems. PUBLIC HEALTH RELEVANCE: Project Narrative Investigation of the unusual 3'-5'nucleotide addition reactions catalyzed by Thg1 family members is of importance to human health, due to the absolute biological requirement for Thg1 activity for tRNAHis function in eukaryotes including humans, the recently demonstrated link between Thg1 overexpression and diabetic nephropathy, and the possibility of discovering novel pathways of mitochondrial 5'-tRNA editing and/or repair, defects in which could contribute to the pathology of human diseases. Moreover, a detailed understanding of the mechanism of Thg1 catalysis in diverse organisms may lead to identification of unique properties of Thg1 homologs from significant human pathogens, such as Plasmodium falciparum and Trichomonas vaginalis, which can be targeted for the development of new antiparasitic or antifungal agents.